The innate immune system is capable of detecting unique microbial components in both the extracellular space and intracellular compartments for induction of immune responses for clearance of pathogens. Recent studies have indicated that multiple cytosolic pathways trigger Type I Interferon (IFN) in response to intracellular DNA and RNA molecules. The exact role Type I IFNs play in the outcome of bacterial infections remains unclear as these cytokines can both prevent and enhance pathogen replication. Chlamydia trachomatis induces a potent Type I IFN response in many different cell types that is dependent on multiple pattern recognition receptors including Toll-like and Nod receptors. However, the regulatory mechanisms underlying Chlamydia-induced Type I IFN responses and the bacterial ligands necessary for this response are poorly understood. The objective of this application is to define the mechanism by which host cells detect Chlamydia infection to induce Type I Interferon responses. My hypothesis is that Chlamydia c-di- nucleotides are detected through cytosolic sensing pathways, which are critical for the host Type I interferon response. We will use murine embryonic fibroblasts (MEFs) stably expressing STING-myc or an Interferon Stimulated Response Element (IRSE)-luciferase reporter to define the signaling pathways that are required for proper STING localization in response to Chlamydia infection and through cytosolic delivery of c-di-AMP. Preliminary results show that the calcium-dependent phospholipase cPLA2 is required for Type I IFN induction in response to both C. trachomatis infection and treatment with c-di-AMP, suggesting that detection of c-di-AMP during Chlamydia infection is required for Type I IFN responses. We plan to test this further by purifying the putative Chlamydia di-adenylate cyclase (DAC) protein CT012 and testing for conversion of radiolabeled ATP to c-di-AMP using thin layer chromatography. To complement this assay I will over express the putative phosphodiesterase recJ in MEFS and screen for the loss of Chlamydia-induced Type I IFN responses. We have assembled a large library of chemically derived Chlamydia mutants and will use this library to screen for bacterial factors that are required for altered Type I IFN responses. We will screen our mutants for induction of Type I IFN using MEFs expressing ISRE-luc and also identify whether these responses correlate with STING translocation. Mutants showing aberrant Type I IFN phenotypes will be sequenced to identify the causative SNPs. The recently characterized genetic exchange with marked strains will allow us to backcross our mutants with wildtype strains to identify a single causative SNP. We will use solid phase extraction on supernatants from EBs and EBs that induce altered Type I IFN responses. The fractionated supernatants will be delivered by digitonin permeabilization to the host cytosol to identify the Type I IFN inducing bioactive molecule. These experiments will significantly advance our understanding of how Chlamydia induces innate immune responses. In addition, we will define the host cellular pathways required for sensing intracellular molecules such as c-di-AMP. PUBLIC HEALTH RELEVANCE: The objective of this application is to define the mechanism by which host cells detect Chlamydia infection to induce Type I Interferon responses. My hypothesis is that Chlamydia c-di-nucleotides are detected through cytosolic sensing pathways, which are critical for the host Type I interferon response.